To date, many expression systems for recombinant proteins have been developed, for various biotechnological applications. Systems for heterologous or homologous gene expression have been established in prokaryotes, yeasts and fungi and in mammalian cells.
Most recombinant proteins produced in yeasts have been expressed using Saccharomyces cerevisiae as the host system. Despite this, several limitations have been detected in the S. cerevisiae system. Examples are product yield, which is usually low, and inefficient secretion (many S. cerevisiae proteins are not found free in the culture medium but rather are retained in the periplasmic space or associated with the cell wall) (Dominguez et al. Int. Microbiol., 1998, vol. 1(2), 131-142). Because of limitations of production in yeast, a lot of interest arose for expression of proteins in bacteria, which are easy to grow in an inexpensive broth and are frequently used to produce recombinant proteins. Among prokaryotic systems, the highest protein levels are usually obtained using recombinant expression in Escherichia coli (E. coli) (Jana & Deb. Appl. Microbiol. Biotechnol., 2005, vol. 67(3), 289-298). However, in E. coli, the most commonly used production strategies are intracellular (in the periplasm or cytoplasm), and therefore involve expensive and often problematic downstream purification processes.
Lactic Acid Bacteria (LAB) are becoming increasingly important as hosts for recombinant expression of heterologous polypeptides in vitro (e.g., U.S. Pat. No. 5,559,007), as well as for in vivo or in situ expression and delivery of antigens and/or therapeutically relevant polypeptides (e.g., WO 97/14806). Heterologous proteins produced in these Gram-positive bacterial hosts can easily be secreted into the medium, thus facilitating their purification as well as their direct delivery to subjects.
Most expression systems can handle very well the expression of one single protein (as a result of one single gene sequence). However, in some cases it is desirable to have an expression system that is capable of expressing multiple proteins or multigenic protein complexes, for example, the in vitro expression of antibodies or protein complexes, but also in vivo or in situ expression and delivery of two or more proteins that have a synergistic effect in a particular disease or the in vivo or in situ expression and delivery of antibodies or functional (multigenic) fragments thereof. In these cases, it is desirable to have the multiple genes that are encoding the desired proteins or antibodies under the control of one promoter, because of the necessity of tight co-regulation of the multiple genes.
The two most common approaches to produce recombinant protein complexes are to perform in vitro reconstitution of individually expressed and purified subunits, or to implement in vivo reconstitution by co-expressing the subunits in an appropriate host (Selleck & Tan, “Recombinant protein complex expression in E. coli”, Curr. Protoc. Protein Sci., 2008, chapter 5:unit 5, 21). Although in vitro reconstitution has been successfully used, the process is tedious (each subunit has to be expressed and purified, and the complex has to be further purified after reconstitution) and reconstitution yields are often low. In contrast, in vivo reconstitution by co-expression offers the benefits of efficiency (only one round of expression and purification) and potentially higher yields and quality of the desired complex (refolding and assembly of the complex take place in the presence of protein folding enzymes in a cellular environment) (Selleck & Tan 2008, supra). In vivo reconstitution has been successfully performed by co-infecting insect cells with baculoviruses expressing individual protein subunits (Tirode et al. Mol. Cell, 1999, vol. 3(1), 87-95), and in bacteria from multiple plasmids (Johnston et al. Protein Expr. Purif., 2000, vol. 20(3), 435-443; McNally et al. Proc. Natl. Acad. Sci. USA, 1988, vol. 85(19), 7270-7273) or from specialized polycistronic plasmids (Henricksen et al. J. Biol. Chem., 1994, vol. 269(15), 11121-11132; Ishiai et al. J. Biol. Chem. 1996, vol. 271(34), 20868-20878; Li et al. Proc. Natl. Acad. Sci. USA, 1997, vol. 94(6), 2278-2283).
General polycistronic expression systems for producing protein complexes in E. coli have been described (Selleck & Tan 2008, supra; Tan. Protein. Expr. Purif., 2001, vol. 21(1), 224-234; Tan et al. Protein Expr. Purif., 2005, vol. 40(2), 385-395). These systems utilize the concept of a translation cassette, comprised of the coding region with requisite START and STOP codons and preceded by translational initiation signals such as the Shine-Dalgarno (SD) sequence and translational enhancers (Tan 2001, Tan et al. 2005, supra). When transcribed into mRNA, the translation cassette contains the necessary and sufficient information for the E. coli translational machinery to initiate and sustain translation of the mRNA into the desired polypeptide (Selleck & Tan 2008, supra).
A bi-cistronic expression vector for interleukin-18 has been described in E. coli, however, the intergenic region between the two genes consisted of a synthetic linker, and is clearly gene-specific as the expression of the caspase-4 was much higher than the expression of ICE. Smolke et al. previously demonstrated that it is possible to differentially control the protein levels encoded by two or more genes in an operon using synthetic intergenic region sequences (Smolke et al. Appl. Environ. Microbiol., 2000, vol. 66(12), 5399-5405; Smolke & Keasling. Biotechnol. Bioeng., 2002, vol. 80(7), 762-776). However, this approach relies on random combinations, and requires the introduction of synthetic sequences into the expression host.
The demand for new and improved antibody production systems has arisen in recent years. Systems for antibody expression have been established in prokaryotes, yeasts and fungi and in mammalian cells. Although single chain and single domain antibodies are easier to produce from bacteria, full-size antibodies generally have higher binding affinities and less risk for formation of neutralizing antibody when injected.
Full-size antibodies can be produced from bacteria (Mazor et al. Nat. Biotechnol., 2007, vol. 25(5), 563-565; Simmons et al. J. Immunol. Methods, 2002, vol. 263(1-2), 133-147). Most reports on recombinant prokaryotic expression describe production of antibody fragments, albeit almost exclusively from E. coli. Although many engineered LAB are capable of correct disulphide bonding, the literature contains only a limited number of examples of antibody-like molecules produced from LAB (Kruger et al. Nature Biotechnology, 2002, vol. 20(7), 702-706; Beninati et al. Nature Biotechnology, 2000, vol. 18(10), 1060-1064; Chancey et al. J. Immunol., 2006, vol. 176(9), 5627-5636; Hultberg et al. BMC Biotechnol., 2007, vol. 7, 58; Yuvaraj et al. Mol. Nutr. Food. Res., 2008, vol. 52(8), 913-920). These reports only describe single chain antibody fragments expressed in Lactobacillus species, Lactococcus lactis and Streptococcus gordonii, and not multigenic, double chain antibody fragments or full-sized antibodies.
Polycistronic expression systems could be crucial in obtaining efficient prokaryotic synthesis and expression of complex proteins such as antibodies. Since the FDA approval in 1986 of Muromonab-CD3, still one of the most potent immunosuppressive drugs available for the management of transplant rejection (Hooks et al. Pharmacotherapy, 1991, vol. 11(1), 26-37), full-size antibodies and antibody fragments have become increasingly important and versatile tools in medicine.
While the current state of the art reveals several examples of polycistronic expression systems in bacterial cells, these are quite limited, highlighting the need for a more efficient system for introducing and expressing multiple genes. Accordingly, there exists a need to provide further sequences which can be favourably used for expression of proteins, preferably heterologous protein expression and even more preferably multiple heterologous protein expression.
In addition to the above, the endeavour to produce higher amounts of recombinant protein, both for direct protein delivery by recombinant microorganisms as well as for bulk protein production and down stream purification, represents a great technological strive. An existing approach to increase production of heterologous proteins is the use of selected strong promoters (see for instance WO 2008/084115). In this approach, proteomic analysis is performed to identify the most abundant endogenous proteins expressed by a microorganism. By use of the genome sequence, the respective genes and promoters can be identified and isolated. These strong promoters (e.g. the Lactococcus lactis hIIA gene promoter, PhIIA) can be positioned in front of a heterologous gene and in this way, high expression can be achieved. However, a level of expression which impairs host physiology may impose a growth burden on the host and results in counter-selection. This intrinsically limits the highest possible expression of any given heterologous protein in an expression host to a certain specific level. This is an especially cumbersome obstacle in the development of chromosomally located expression units.
The issue of counter-selection is traditionally addressed by the provision of selection markers. Indeed, positive or negative selection, e.g. by providing antibiotic resistance genes, can prevent loss of the introduced heterologous gene. Alternatively, or in addition to the use of selection markers, inducible gene expression systems may be employed, which allow for uncoupling propagation of the host and expression of the heterologous protein, thereby preventing possible counter-selection during the propagation phase when the heterologous gene is not expressed. In this context, EP0569604 describes an inducible expression system in Streptococcus thermophilus in which a heterologous gene is obligatory positioned 5′ to the LacZ gene. In this way, expression of the heterologous gene is not only inducible but in addition maintenance of the heterologous gene is also selected for by growing the bacteria in their natural habitat, milk, with lactose as carbon source, which requires the expression of the LacZ gene.
It is clear that the systems for heterologous gene expression described above are limited in application. For instance, the use of selection markers, such as antibiotic resistance genes, is not readily tolerated for applications in food production or in pharmaceutical applications. Further, limitation to growing in the natural habitat or to using the carbon source from the natural habitat for growth significantly reduces the versatility of any system for heterologous gene expression. Also, the use of inducible systems is inherently dependent on the growth conditions of the host, such that defined culture media, to which an inducer is to be added, are needed to ensure expression of the heterologous protein.
Accordingly, there also exists a need in the art to increase heterologous protein expression; and sequences, cloning systems and strategies are needed which can achieve high expression levels in order to obtain sufficient amounts of expressed heterologous proteins in industrial and/or therapeutic settings, while at the same time being versatile and widely applicable under a variety of different conditions. In these settings it would also be particularly useful to obtain expression of multiple proteins, each having its own biological activity and therapeutic effect.